The C. elegans flr-3(ut9) mutation is a loss-of-function insertion within the drl-1 locus

The genes encoding the mitogen-activated protein kinases DRL-1 and FLR-4 are required for growth and lipid homeostasis in C. elegans . Interestingly, the flr-3 ( ut9 ) mutant, which was previously isolated in a forward genetic screen for mutations that confer fluoride resistance, phenocopies the drl-1 and flr-4 loss-of-function mutants; however, the genetic identity of flr-3 is unknown. Through whole genome sequencing, we found that the flr-3 ( ut9 ) mutation is an insertion in the drl-1 locus and disrupts drl-1 gene function, resulting in dramatic growth defects and impaired vitellogenin production.

), or flr-3(ut9) animals.H) A schematic showing the genetic lesions within the drl-1 locus and the primer pairs used to genotype the ut9 allele.Amplification of the I) wild-type or J) ut9 drl-1 alleles by PCR in wild-type, drl-1(rhd109), and flr-3(ut9) animals using the indicated primer pairs.PCR products were resolved by agarose gel electrophoresis.All strains shown here carry the Pvit-3::mCherry reporter.

Description
In C. elegans, vitellogenesis is the process by which triglycerides and cholesteryl esters are transported from intestinal cells to the germline in the form of lipoprotein particles to support reproduction and embryonic development (Perez and Lehner 2019).Adult-specific expression of the vitellogenin (VIT) proteins, which is necessary for lipoprotein assembly, is highly regulated and can be tuned by developmental, reproductive, and environmental inputs (DePina et al. 2011;Dowen et al. 2016;Seah et al. 2016;Dowen 2019).
We previously identified two genes, drl-1 and flr-4, that are required for proper initiation of vitellogenesis at adulthood (Torzone et al. 2023).Both drl-1 and flr-4 encode serine/threonine mitogen-activated protein kinases (MAPK) that are orthologues of mammalian MEKK3 (Take- Uchi et al. 1998;Chamoli et al. 2014).In addition to vitellogenesis defects, mutation of either gene results in slowed development and a severe reduction in body size (Torzone et al. 2023).Intriguingly, the flr-4 mutation was isolated in a forward genetic screen for fluoride resistant mutants along with the flr-3(ut9) mutant, which is also slow-growing but is not allelic to flr-4 (Katsura et al. 1994).
To identify the ut9 mutation, we performed whole genome sequencing of a pool of F2 recombinants after backcrossing flr-3(ut9) to wild-type Pvit-3::mCherry animals (Doitsidou et al. 2010).This approach led to the discovery of an insertion within exon 7 of the drl-1 coding sequence (Figure 1H).Notably, the 5' and 3' arms of the insertion match sequences within known Tc1 transposons found throughout the genome, suggesting that the ut9 allele is likely a Tc1 insertion (see Extended Data for sequences).Because of the repetitive nature and the size of the insertion, we were unable to amplify the full insertion by PCR; and thus, the full sequence of the insertion remains unknown.Using a primer that is positioned within the 3' end of the insert, we verified that the insertion was specific to the flr-3(ut9) strain using PCR (Figure H-J).Moreover, these genotyping results corroborate our whole genome sequencing approach and suggest that the ut9 mutation is likely a Tc1 insertion within the drl-1 locus.Thus, we propose renaming the flr-3(ut9) mutation to drl-1(ut9).
In conclusion, our results indicate that the drl-1(ut9) mutation impairs vitellogenesis, developmental rate, and body size to a similar degree as the drl-1(rhd109) frameshift mutation, which we have previously characterized as a strong loss-of-function allele (Torzone et al. 2023).It is likely that both alleles are null alleles given the size of the ut9 insertion and where it is positioned in the coding region of the drl-1 gene.Therefore, while loss of drl-1 produces severe phenotypes it is unlikely to be an essential gene.This study revealed the identity of the flr-3(ut9) mutation and provides a new tool to investigate the DRL-1/FLR-4 MAPK pathway.

Methods
C. elegans strains and maintenance C. elegans strains were cultured at 20ºC on Nematode Growth Media (NGM) plates seeded with E. coli OP50 as previously described (Brenner 1974).The JC53 strain was obtained from the Caenorhabditis Genetics Center.

Fluorescent reporter imaging
L4 animals carrying the Pvit-3::mCherry reporter were picked to fresh E. coli OP50 plates and 24 hours later the animals were 12/5/2023 -Open Access mounted on a 2% agarose pad with 25mM levamisole.The day 1 adult animals were imaged with a Nikon SMZ-18 Stereo microscope equipped with a DS-Qi2 monochrome camera.The mCherry fluorescence was quantified and analyzed as previously described (Torzone et al. 2023).

Growth rate assay
Animals were grown for two generations on NGM plates seeded with E. coli OP50 prior to being assayed.Approximately 100 eggs from each strain were picked to the bacterial lawn of a new NGM plate.Animals were scored every 24 hours for 7 days and gravid adults were picked off at each timepoint and recorded as having reached adulthood.
Body size assay L4 animals grown for two generations on NGM plates were picked to fresh plates and imaged 24 hours later using a Nikon SMZ-18 Stereo microscope equipped with a DS-Qi2 monochrome camera.Individual animals were traced using the ImageJ2 v2.3.0 software (Rueden et al. 2017) and the two-dimensional outline was used to calculate the body size of each animal in mm 2 .

Whole genome sequencing and genotyping
The flr-3(ut9) strain was crossed to DLS537 and F2 recombinants that displayed reduced reporter expression and slow growth were picked and singled to individual NGM plates.Animals from approximately 50 starved F2 plates were pooled (Doitsidou et al. 2010), the genomic DNA was harvested using the Gentra Puregene Tissue Kit (Qiagen), and the gDNA-Seq libraries were prepared and sequenced using the Illumina NovaSeq platform (Novogene Inc.).The candidate mutations were identified using in-house scripts (Minevich et al. 2012).Sequencing reads surrounding the insertion site were manually inspected to identify the exact location of the insertion and the sequence of the insertion was manually constructed using overlapping reads.Because the 5' and 3' ends of the insertion are identical to other Tc1 transposon sequences found throughout the genome, we were unable to precisely determine the full sequence of the ut9 insertion from our whole genome sequencing data.Specific amplification of the ut9 allele was carried out by PCR using the forward primer 5'-GTCATTTCCTTGCAACCTCG-3' and the universal reverse primer 5'-CCACATCAGCGTGATATCTG-3', while the wild-type allele was amplified with the forward primer 5'-CCAGAAAATCGACCATCTGC-3' and the universal reverse primer.The PCR products were resolved by agarose gel electrophoresis and visualized with ethidium bromide.

Statistics
All statistical analyses (one-way ANOVA with a Bonferroni's multiple comparisons correction) were performed in Prism v9.